Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures

Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we use a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes. These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.


Introduction
Membranes form barriers that separate cells from their environment and separate diverse subcellular compartments so that they can carry out distinct functions within cells 1 .
Membranes are also highly dynamic, undergoing fusion and fission events during endocytosis, ectocytosis, and cytokinesis 2,3 . As membrane bilayers are less than 10 nm in diameter, light microscopic techniques struggle to distinguish neighbouring membranes or to tell when membranes have fused to have a closed topology 4 . Therefore, techniques that allow specific labelling of distinct structures are required to visualize membrane dynamics and probe membrane topology in living cells.
Fluorescent reporters binding specific phosphatidylinositol species are popular for studying membrane dynamics 5 , but these lipids are often present on multiple structures, making it hard to distinguish individual organelles. One example is during phagocytosis, when cellular debris or cell corpses are engulfed by the plasma membrane 6 . Both the corpse and engulfing cell plasma membranes contain the same phosphatidylinositol species, making it challenging to distinguish these membranes in living cells. Electron microscopy and super-resolution light microscopy can visualize the few tens of nm that separate the phagosome membrane from the corpse membrane 7 , but these techniques rely on fixation, which makes it challenging to study dynamics. Another example is extracellular vesicles released from cells. The content of vesicles that bud from the plasma membrane by ectocytosis is similar to the membrane and associated cytoplasm that they originate from 8 , which makes it hard to distinguish released vesicles from the plasma membrane of neighbouring cells. The lack of specific markers for extracellular vesicles using conventional reporters has limited our understanding of their cell biology 9 . Thus, new approaches are needed to label specific membranes.
In order to develop reporters that visualize specific membrane structures, we repurposed the cell's endogenous machinery for selective degradation. We were inspired by protease protection assays using exogenous proteases 10 , but wanted to establish an in vivo system that did not require detergent-mediated permeabilization of the plasma membrane.
Degrons are degradation motifs that target specific proteins for ubiquitination and degradation, which has led to degron-tagging being used as an alternative loss-of-function approach to RNA interference or genetic knockouts 11 . Degrons recruit ubiquitin ligases to polyubiquitinate target proteins, resulting in the proteasomal degradation of cytosolic targets or the lysosomal degradation of transmembrane targets 12 . Rather than using degron tags for a loss-of-function technique, we used the degradation of degron-tagged reporters in the cytosol to specifically label certain cells, cell fragments, organelles, and vesicles.
To test endogenous degrons, we primarily used the zinc finger 1 (ZF1) degron from the elongin C subunit of an ECS ubiquitin ligase complex 13 . ZIF-1 is expressed in sequential sets of differentiating somatic cells 14 , resulting in a stereotyped pattern of degradation in developing embryos 13 (Fig. 1a). Fusing the ZF1 degron to a target protein results in degradation within 30 to 45 min of ZIF-1 expression in both embryonic and adult tissues 15 . As an alternate approach, we used the C-terminal phosphodegrons (CTPD) from the C. elegans OMA-1 protein 16 . Two threonines in the C-terminus of OMA-1 are phosphorylated after fertilization, leading to recognition of OMA-1 by multiple SCF ubiquitin ligase complexes and rapid proteasomal degradation in embryos at the end of the 1-cell stage 16,17 (Fig. 1b). We also used a heterologous degron in mammalian cells, the auxin-inducible degron (AID) from plants 18 . The 68 amino acid AID motif from IAA17 is recognized by the F-Box protein TIR1 in the presence of the auxin family of plant hormones 19 . Auxins are cell permeable and TIR1 is able to become part of endogenous SCF ubiquitin ligase complexes in model systems from yeast to mammals in order to ubiquitinate AID-tagged proteins 18 . The AID system is thus a three-component system, allowing temporal control of degradation by addition of auxin hormone and spatial control of degradation by the expression of TIR1 in different cells (Fig. 1c).
Here, we show that degron-tagged reporters separated from the ubiquitin ligase complex by intervening membranes are no longer accessible to ubiquitination and degradation in C. elegans embryos or mammalian cells. This results in background-free labelling of specific cells, organelles, and vesicles. This improvement in the signal-to-noise ratio enabled the visualization of extracellular vesicles in vivo, the long-term tracking of individual phagosomes, as well as distinguishing a corpse plasma membrane from the engulfing phagosome membrane in vivo. In addition, degron-tagging allowed us to measure the timing of nuclear envelope breakdown and abscission during cell division. Degron-tagged reporters thus provide a convenient method for investigating in vivo dynamics from the level of proteins to cells.

Results
To determine whether degron-tagged reporters would be useful for cell biological approaches, we tested whether an endogenous degradation system was capable of degrading abundant reporter proteins and examined whether the increased proteasomal load had negative effects on cells. We tagged a membrane-binding domain, the PH domain of rat PLC1∂1, with the ZF1 degron from C. elegans PIE-1 and expressed it in worm embryos. Similar to an mCherry-tagged PH reporter (Fig. 2a), the cytosolic mCh::PH::ZF1 reporter initially localizes to the plasma membrane (Fig. 2d). Thus, the degron tag does not disrupt the normal localization of the reporter.
ZF1-mediated degradation begins in anterior somatic cells at the 4-cell stage, due to the onset of expression of the ubiquitin ligase adaptor protein ZIF-1 14 . While mCh::PH fluorescence persists in developing embryos (Fig. 2b-c), mCh::PH::ZF1 is progressively degraded, starting with anterior somatic cells ( Fig. 2e-g, video 1). ZIF-1 is not expressed in the germ lineage, resulting in persistent fluorescence in a couple of posterior cells (Fig. 2e-f). ZIF-1 expression also does not occur in two small cell corpses born at the anterior side of the embryo during meiosis 14,20 . These polar bodies maintain mCh::PH::ZF1 fluorescence (arrowheads in Fig. 2). Thus, the degron tag led to rapid degradation of a highly-expressed, exogenous reporter in cells where the ligase adaptor was expressed and the reporter could therefore be ubiquitinated.
As ZIF-1 has a number of known targets whose proteasomal degradation is important for embryonic development 13 , we tested whether the expression of ZF1-tagged reporters disrupted development. Stable transgenic strains expressing various ZF1-tagged reporters were fertile and had similar numbers of viable progeny that did not show developmental delays (Fig.   S1a). In fact, most ZF1-tagged reporter embryos developed significantly faster than corresponding reporter strains without the degron (Fig. S1b) and were less likely to show overexpression defects ( Fig. S1c-f). This suggests that degron reporters can be tolerated better than other overexpressed reporters and do not generally disrupt development.

Degron reporters specifically label released extracellular vesicles
In addition to labelling the plasma membrane, mCh::PH and mCh::PH::ZF1 labelled intracellular vesicles (arrows in Fig. 2), some of which maintained their fluorescence in the mCh::PH::ZF1 strain (video 1). As mCh::PH::ZF1 on the cytosolic face of vesicles would be accessible for ubiquitination and proteasomal degradation, the persistence of the degron reporter suggests that it is protected from degradation by intervening membranes. We hypothesized that the PH::ZF1 reporter persisted in extracellular vesicles (EV) or other cell debris that are taken up by the cell during endocytosis.
To test whether the degron-tagged PH reporter could be used to specifically label and track EVs released in vivo, we examined microvesicles. Microvesicles are 90-500 nm vesicles that arise from plasma membrane budding, aka ectocytosis 8 . In wild type embryos, microvesicles are difficult to detect due to their low abundance and proximity to the plasma membrane. Microvesicle budding is normally inhibited by the TAT-5 lipid flippase, resulting in continuous microvesicle release when tat-5 is knocked down, as demonstrated by electron tomography 21 . In mCh::PH embryos, microvesicle overproduction is visible as thickened membrane labelling between tat-5 knockdown cells (Fig. 2h-j) in comparison to control embryos ( Fig. 2a-c). However, small patches of microvesicles are difficult to detect over the background of the plasma membrane fluorescence. In contrast, released microvesicles are clearly visible after tat-5 knockdown using the mCh::PH::ZF1 reporter (Fig. 2l-m) 2 , due to proteasomal degradation of the plasma membrane label (Fig. 2n). Released EVs are then visible floating between the embryo and the eggshell (Video 1). Thus, degron tagging a general plasma membrane reporter reveals microvesicles and their movement in vivo.
To determine whether this was a specific feature of the ZF1 degron or the ECS ubiquitin ligase, we tested whether another degron could be used with SCF ubiquitin ligases to label EVs.
We tagged the PH reporter with a C-terminal fragment of OMA-1 (aa219-378) containing two phosphorylation sites important for degradation 16 , which we named the C-terminal phosphodegrons (CTPD) (Fig. 3d). Early during the 1-cell stage, mCh::PH::CTPD localized brightly to the plasma membrane, but began to be degraded during the first mitotic division (Fig. 3a, Video 2). Degradation was transient and some mCh::PH::CTPD persisted on the plasma membrane after the 2-cell stage (Fig. 3c). Even with partial degradation, microvesicles could readily be observed with mCh::PH::CTPD after tat-5 RNAi treatment (Fig. 3b, Video 2).
Thus, even a partial loss of plasma membrane signal enhanced visualization of EVs.
We also tested whether it was possible to label EVs by degron-tagging transmembrane proteins. In contrast to the proteasomal degradation of cytosolic proteins, ubiquitination of transmembrane proteins leads to endocytosis and lysosomal degradation 12 . The syntaxin SYX-4 is a single-pass transmembrane protein that localizes to the plasma membrane and endocytic vesicles ( Fig. S2a-c) 22 . We tagged the cytosolic domain of SYX-4 with the ZF1 degron to make it accessible to ZIF-1. Degron-tagged GFP::ZF1::SYX-4 localizes normally before the onset of ZIF-1 expression (Fig. S2d), after which GFP::ZF1::SYX-4 accumulates in intracellular vesicles and is lost from the plasma membrane (Fig. S2e). These vesicles eventually disappear from ZIF-1-expressing cells (Fig. S2f), consistent with ubiquitin-driven endocytosis and lysosomal degradation (Fig. S2i). To test whether lysosomal degradation of a transmembrane protein can label EVs, we treated the GFP::ZF1::SYX-4 reporter strain with tat-5 RNAi to induce microvesicle release. Similar to the degron-tagged PH reporter ( Fig. 2k-n), GFP::ZF1::SYX-4 accumulates around cells after tat-5 knockdown (Fig. S2g-h) 21 . Thus, both membrane-associated and transmembrane proteins can be tagged with degrons to specifically label EVs.

Degron protection assay reveals topology of membrane-associated proteins
We next tested whether degron tagging could reveal insights into protein topology.
Clathrin is enriched at the cell surface after tat-5 knockdown 21 , but it was unclear whether this was due to increased clathrin inside the plasma membrane or due to the release of clathrin in EVs that accumulated next to the plasma membrane (Fig. 4f). Both possibilities were plausible, as clathrin-binding proteins are increased at the plasma membrane after tat-5 knockdown, including ESCRT proteins 21 , and as clathrin was found in purified Drosophila and mammalian EVs 23,24 . Therefore, we asked whether a degron-tagged clathrin heavy chain reporter would be protected from degradation inside EVs or be accessible to degradation at the cell cortex.
This demonstrates that the increased clathrin signal is due to association with the plasma membrane and not due to clathrin trapped within EVs. Thus, degron-tagged reporters reveal whether a protein is inside or outside the plasma membrane.

Degron reporters enable tracking of phagocytosed cargo
To test whether the specific labelling by degron tags facilitates long-term tracking, we observed the two polar bodies in which ZF1 degradation does not occur ( Fig. 2d-f). As polar bodies are dying cells, they have a nucleus and can be tagged with chromosome reporters like histone H2B, in addition to the PH domain 20 . Both polar bodies are initially found on the anterior surface of the embryo (Fig. 5a, d). The first polar body is trapped in the eggshell 25 , while the second polar body (2PB) is phagocytosed by an anterior cell 20 . Because mCh::H2B labels all nuclei in the embryo, it can be challenging to track the 2PB phagosome among the many dividing nuclei (Fig. 5b-c, Video 3). In contrast, the degron-tagged ZF1::mCh::H2B reporter disappears sequentially from somatic nuclei, leaving the two polar bodies as the only fluorescent structures on the anterior half of the embryo (Fig. 5e, Video 3). This confirms that the intervening cell corpse and phagosome membranes protect ZF1-tagged proteins from proteasomal degradation (Fig. 5g). Improving the signal-to-noise ratio with degron-tagged reporters also improves automated tracking of the 2PB by removing overlapping traces (Fig.   5c, f). Tubulation of the 2PB phagosome into small vesicles can also be followed with degrontagged reporters (video 1, left) 20 . Thus, degron-tagged reporters reveal organelle dynamics and facilitate tracking by removing background labelling.

Degron reporters reveal plasma membrane topology and dynamics
Degron-tagged reporters also allow the specific labelling of a single membrane or surface of a membrane. The close proximity of the corpse plasma membrane and the engulfing phagosome membrane make it difficult to distinguish their signals using fluorescence microscopy 26,27 , unless cells have distinct transcriptional programs, i.e. neuronal corpses engulfed by non-neuronal cells. Using degron reporters, this can be achieved even with sister cells in vivo by degrading the reporter localized to the phagosome membrane, leaving the corpse membrane preferentially labelled (Fig. 6d). For example, as polar bodies do not enter mitosis, their plasma membranes remain brightly labelled after mCh::PH::CTPD degradation occurs in the embryo (Fig. 6a). After phagocytosis, the 2PB membrane appears as a bright hollow sphere in a weakly labelled cell using the mCh::PH::CTPD reporter (Fig. 6b). In order to degrade or recycle corpse contents, the plasma membrane of engulfed corpses must be disrupted within the safety of the phagosome membrane 28 . 2PB membrane breakdown can be visualized using the mCh::PH::CTPD reporter, which is seen dispersing throughout the phagosome lumen (Fig.   6c). Similar results were found using the mCh::PH::ZF1 reporter (Video 1) 20 . Thus, by depleting fluorescent reporters from membrane surfaces facing the cytosol, degron-tagging enables examination of specific membranes and their dynamics.

Degron protection assays reveal nuclear membrane topology and dynamics
We next asked whether degron-tagged reporters can be used to assess nuclear topology.
Other components of the ECS ubiquitin ligase complex are found in both the cytosol and nucleus, but the ZIF-1 ligase adaptor appears cytosolic 29 . To test whether ZIF-1 degradation was restricted to the cytosol (Fig. 7a), we tagged the nuclear lamin LMN-1 with the ZF1 degron to examine the dynamics of the nuclear cortex during cell division 30 . Prior to the onset of ZIF-1 expression, the fluorescence intensity of the mKate2::ZF1::LMN-1 reporter is comparable between the anterior and posterior nuclei (Fig. 7c). After ZIF-1 expression begins in the two anterior daughter cells, the interphase levels of mKate2::ZF1::LMN-1 gradually drop in comparison to posterior cells (Fig. 7d, g), suggesting that some degradation occurs despite the intact nuclear envelope. However, the degradation of the mKate2::ZF1::LMN-1 occurred faster during mitosis ( Fig. 7e-g, video 4). The nuclear envelope breaks down (NEBD) during mitosis for chromosome segregation 31 , which would allow cytosolic ZIF-1 to target ZF1-tagged nuclear proteins (Fig. 7b).
To confirm that loss of fluorescence was due to ZIF-1-mediated degradation and not due to morphological changes in the nuclear envelope during the cell cycle, we treated reporter embryos with zif-1 RNAi to inhibit degradation. The fluorescence of mKate2::ZF1::LMN-1 fluctuated as cells divided (Fig. 7g), but fluorescence persisted in anterior cells (Fig. 7i). We normalized the control curve to the zif-1 curve to remove changes due to nuclear morphology, revealing 4-6 times faster degradation during NEBD (Fig. 7h). Thus, although a pool of mKate2::ZF1::LMN-1 is accessible to ZIF-1 during interphase, mKate2::ZF1::LMN-1 is largely protected from proteasomal degradation by the nuclear envelope (Fig. 7a). After treating mKate2::ZF1::LMN-1 embryos with zif-1 RNAi, we noticed an increase in nuclear morphology defects (Fig. S1c). However, the mKate2::ZF1::LMN-1 reporter did not result in other defects typical of LMN-1 overexpression, even when zif-1 was knocked down (Fig. S1d-f). Therefore, degron-tagging provides probes for membrane topology that can investigate the dynamics of nuclear envelope breakdown. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint As LMN-1 is largely immobile in the nuclear lamina 32 , the observation that some degradation occurred during interphase raised the possibility that ubiquitination of the degrontagged reporter was altering LMN-1 dynamics. To determine whether mKate2::ZF1::LMN-1 became mobile after ZIF-1 expression, we performed fluorescence recovery after photobleaching (FRAP) experiments in an anterior daughter nucleus (Fig. S3a). There was no significant recovery of mKate2::ZF1::LMN-1 fluorescence during interphase (Fig. S3b), suggesting that the mobility of LMN-1 was unchanged by ubiquitination. Thus, quantification of degron-tagged reporters can be a tool to examine protein dynamics in addition to organelle dynamics.

The localization of ubiquitin ligase adaptors determines the localization of degradation
We next tested whether protection by internal membranes was a specific feature of ZIF-1 and ECS ubiquitin ligases or whether the nuclear envelope could also protect targets of SCF ubiquitin ligases from degradation. As SCF ubiquitin ligase complexes are found in the nucleus and cytosol, we modified the TIR1 ligase adaptor from plants with a nuclear export signal (NES) and expressed NES-TIR1 in HeLa cells that also expressed a lamin A reporter tagged with a minimized AID (mAID) degron 33 . During interphase, little Venus-mAID-LMNA fluorescence was lost when TIR1 was restricted to the cytosol (Fig. 8a, c, f, Video 5). After cells entered mitosis and the nuclear envelope broke down (NEBD), NES-TIR1 caused rapid degradation of Venus-mAID-LMNA ( Fig. 8b-c, f, Video 6). Thus, degron protection assays can also be used in mammalian cells.
To confirm that protection from degradation was due to the localization of the ligase adaptor and not due to a protective effect of the nuclear lamina, we co-expressed NLS-TIR1, which has a nuclear localization signal (NLS), with NES-TIR1 33 . When TIR1 was localized to both the nucleus and the cytosol, Venus-mAID-LMNA was rapidly degraded during interphase ( Fig. 8d-e, g, Video 7). The velocity of interphase degradation by NLS-TIR1 was not significantly different from mitotic degradation by NES-TIR1 (Fig. 8h), indicating that LMNA was not protected from ubiquitination by integration into the nuclear lamina. These results demonstrate that the localization of the ligase adaptor can determine the localization of degradation, providing a strategy to target one pool of a reporter protein for degradation.

Degron reporters reveal membrane topology during abscission
In order to understand how degrons in restricted spaces can be ubiquitinated and degraded, we applied degron-tagged reporters to the process of abscission. During cell division, the actomyosin furrow closes around the spindle midbody to form a narrow intercellular bridge 34 . Both sides of the bridge are cleaved during abscission to release a ~1 µm extracellular vesicle called the midbody remnant, which is later phagocytosed (Fig. 9h) 35 . The intercellular . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint bridge no longer permits diffusion between cells ~4 minutes after furrow ingression 34 , but the first cut for abscission does not occur until ~10 minutes after furrow ingression 36 . Therefore, we asked when actomyosin accumulated in the intercellular bridge was accessible for proteasomal degradation. We tagged non-muscle myosin (NMY-2) with the ZF1 degron and measured the fluorescence intensity of NMY-2::GFP::ZF1 in the bridge between the anterior daughter cells (Fig. 9a) 35 . Degradation of cytoplasmic NMY-2::GFP::ZF1 was first visible 8 ± 1 minutes after furrow ingression. NMY-2::GFP::ZF1 in the bridge showed a small but significant decline for the next 2 minutes (Fig. 9g), suggesting that NMY-2 is normally able to diffuse out of the bridge up to 10 minutes after furrow ingression. Subsequently, NMY-2::GFP::ZF1 in the bridge was protected from proteasomal degradation (Fig. 9b, g), suggesting that either a diffusion barrier had formed or abscission had occurred. Thus, using degron reporters and light microscopy on living embryos, we could confirm the timing of abscission estimated from electron microscopy data from fixed embryos.
In contrast to control embryos where NMY-2::GFP::ZF1 fluorescence persisted through midbody release and phagocytosis (Fig. 9b), NMY-2::GFP::ZF1 fluorescence intensity continued to drop significantly in the bridge of both tsg-101 and unc-59 mutants (Fig. 9d, f-g), and this drop was dependent on ZIF-1 expression 35 . Phagocytosis of the midbody remnant was also delayed in both tsg-101 and unc-59 mutants (Fig. 9d, f) 34,35 , consistent with a delay in abscission. These findings demonstrate that NMY-2 is able to diffuse out of the bridge and be degraded when abscission is delayed (Fig. 9i). No defect was detected for tsg-101 knockdown using a dextran diffusion assay 34 , demonstrating the high sensitivity of the degron protection assay for detecting abscission defects.

Single membrane bilayers are sufficient to protect proteins from degron-mediated degradation
As all of the model systems we examined involved two membrane bilayers between the ubiquitin ligase and the degron-tagged reporter (nucleus, extracellular vesicle, phagocytosed cell debris), we tested whether a single membrane bilayer would protect a reporter from degradation. We expressed a degron-tagged reporter in the secretory pathway and maintained it in the endoplasmic reticulum (ER) using a C-terminal KDEL sequence 37 (Fig.   10d). The ss::TagRFP-T::ZF1::KDEL reporter localized to the ER, similar to established reporters ( Fig. 10a-b). After ZIF-1 expression began in anterior cells, ss::TagRFP-T::ZF1::KDEL persisted with no measurable loss of fluorescence ( Fig. 10b-c). Thus, a single . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint membrane bilayer protects degron-tagged reporters from degradation, confirming that degron protection assays can be applied to intracellular organelles.

Degron-tagged reporters did not result in degradation of binding partners
Ligase adaptors can target proteins for ubiquitination through intermediary binding partners, including nanobodies 33 , which raised the possibility that degron-tagged reporters would lead to the degradation of untagged binding partners. This could be especially relevant when the reporter protein assembles into larger complexes, such as histones or the nuclear lamina, or dimerizes, such as NMY-2. To test whether degron-tagged proteins lead to the degradation of untagged proteins, we generated strains expressing fluorescent reporter proteins for H2B, LMN-1, and NMY-2 both with and without the ZF1 degron tag. We measured the intensity of fluorescence for each reporter and found that although the ZF1-tagged reporter degraded, the reporter without the degron did not show significant changes in comparison to a strain that did not express any degron-tagged reporter (Fig. S4). Thus, degron-tagging does not generally lead to degradation of protein complexes.

Discussion
In summary, degron-mediated degradation is more than a loss-of-function technique; it is a powerful tool to study dynamics from the level of proteins to organelles to cells. By removing cytoplasmic fluorescence, degron tags improve the visibility of extracellular, luminal, or nuclear reporters and enable long-term tracking. Degron-tagged reporters reveal insights on an epifluorescence microscope that are typically limited to super-resolution or electron microscopy on fixed samples. Our studies have focused on structures that are protected by membrane bilayers, but this approach should work for any structure resistant to ubiquitination or diffusion. Thus, degron-tagging is an important addition to the cell biologist's toolbox.
As degron tags are widely used in cell extracts, cell culture and in vivo, this approach can visualize structures in many systems. We started with endogenous degrons in C. elegans embryos for their simplicity, only requiring expression of a degron-tagged reporter.
Heterologous expression of ZIF-1 in worms or zebrafish can also degrade ZF1-tagged proteins and could be adapted to more systems 15,38 . Fusing an anti-GFP nanobody to endogenous ubiquitin ligase adaptors like ZIF-1 enables spatial control of degradation of GFP-tagged proteins in C. elegans, Drosophila, plants, and zebrafish [38][39][40][41] , which allows existing GFP-tagged reporters to be used for degron protection assays. Heterologous expression of the auxininducible degron (AID) and TIR1 ligase adaptor is also used in various animal models 18,33,42 .
We used AID in mammalian cells to demonstrate that altering the localization of the ubiquitin ligase adaptor is sufficient to target degradation to the nucleoplasm or cytoplasm, consistent with observations on endogenous ligase adaptors 43 . Thus, the spatial control of degradation . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint enabled by heterologous expression of ligase adaptors refers not only to expression in specific cell types, but also to specific organelles. As different ubiquitin ligases are found in different cellular compartments 44 , the choice of degron-ligase pair can enable degron protection assays in a range of organelles. For example, the Ab-SPOP/FP system uses a BCR ubiquitin ligase complex including Cul3 to specifically target GFP-tagged proteins in the nucleus for degradation 38,45 , but Cul3 also localizes to the Golgi. Furthermore, ubiquitin-mediated degradation can be regulated by small molecule drugs, temperature, or light 11 , offering many modalities to control the timing and localization of protein degradation. Thus, degron protection assays can readily probe cell biology in many model systems.
To avoid loss-of-function effects from induced degradation of degron-tagged proteins, our studies were performed with isolated protein domains or in the background of the untagged endogenous protein. Still, degradation of an overexpressed degron-tagged protein could alter some processes. We found that strains expressing degron-tagged fluorescent reporters were healthier than strains expressing fluorescent-tagged reporters. In fact, we only observed effects on nuclear morphology after inhibiting degron-mediated degradation of a LMN-1 reporter (Fig.   S1c). Regardless, degrons like CTPD or AID that lead to partial or reversible degradation may be advantageous to avoid loss-of-function effects. In addition, it is important to verify that the degron tag does not alter the protein or process under study, similar to other protein tags. We did not observe changes to protein dynamics after degron tagging, with the expected exception of induced endocytosis of a transmembrane protein. Thus, degron tags are useful for probing the topology of transmembrane proteins, but not necessarily for studying their intracellular trafficking.
We used degron tags to label and track structures for which conventional reporters are insufficient. For example, extracellular vesicles (EV) are typically detected by the tetraspanin proteins on their surface, but tetraspanin content is heterogeneous among EV subpopulations 46 .
By degron-tagging membrane-associated or transmembrane proteins, we were able to specifically label EVs in vivo, which has proven to be a valuable tool to screen for new regulators of EV budding 2 . Although our PH::ZF1 reporter is likely to favour plasma membrane-derived EVs (microvesicles), it is possible to target endosome-derived EVs (exosomes) by degron-tagging proteins associated with the endosome surface. Alternatively, exosomes may also be labelled by degron-tagging transmembrane proteins, given that ubiquitination drives endocytosis of transmembrane proteins (Fig. S2i). Degron-tagging reporters found at both the plasma membrane and endosome surface, such as actin-binding domains or abundant proteins like GAPDH, should label both microvesicles and exosomes 47 .
Thus, degron-tagged reporters are uniquely able to specifically label EVs, enabling in vivo tracking and functional studies. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint Wide-field light microscopy is normally limited to detecting structures that are >200 nm away from each other 48 . At this resolution, neighbouring membranes cannot be distinguished. In addition to specifically labelling extracellular vesicles next to the plasma membrane, we showed that degron-tagged reporters could distinguish the cargo corpse membrane from the engulfing phagosome membrane. This enabled the visualization of corpse membrane dynamics during phagolysosomal clearance using wide-field microscopy 20 . These reporters enable the precise staging of phagosomes for approaches such as correlative light and electron microscopy (CLEM) 49 , which can be used to determine the ultrastructure of membrane breakdown during phagolysosomal clearance. Therefore, degron-tagging is a useful tool to reveal novel insights into organelle dynamics in addition to the long-term tracking of specific cells in vivo.
Using a degron-tagged reporter, we were able to measure early changes in the topology of the nuclear envelope during cell division as well as in the restricted space of the intercellular bridge, which was previously only possible using electron microscopy 36 . We were also able to detect defects in abscission after knocking down an ESCRT subunit 35 , which were not visible using cytosolic diffusion assays 34 . Degron protection is therefore a sensitive tool to study the topology of membranes while they undergo fusion or fission.
Degron protection also revealed protein dynamics and topology. Knocking down septins or TSG-101 led to distinct rates of degron-mediated degradation of the myosin reporter, which could indicate that the intercellular bridge is open to differing degrees in these mutants, resulting in different rates of diffusion out of the bridge. We also found that a pool of the degron-tagged lamin reporter underwent degradation during interphase. This may indicate that an undetected level of ZIF-1 is in the nucleus, which ubiquitinates the ZF1 degron during interphase for degradation by the nuclear proteasome 50 . Alternatively, this may be due to undetected mobility of nuclear lamins into the cytosol during interphase. As cytosolic ligases are known to degrade proteins exported from the ER, while ER contents are protected from ECS-or SCF-mediated degradation (Fig. 10) 51 , degron-tagged reporters may also be used to reveal information on protein import/export across organelle membranes. As degradation of a reporter depends on its orientation as well as its location within the cell, degron protection assays can be applied to determining the topology of transmembrane proteins (type I/II) by testing whether they are accessible to degron-induced endocytosis and degradation, similar to our observations with SYX-4. Similarly, degron protection assays can distinguish cytosolic from luminal proteins. In summary, degron-tagged reporters improve the signal-to-noise ratio, reveal super-resolution insights on a standard microscope, and provide insights into localization and dynamics from the level of cells to proteins.

Competing Interests
The authors declare no competing interests.
. CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint

Worm strains and maintenance
Caenorhabditis elegans strains were maintained according to standard protocol at room temperature or 25˚C 52 . For a list of strains used in this study, see Table S1. unc-59 loss-offunction mutant embryos were generated by feeding unc-59 RNAi to the WEH132 strain bearing a hypomorphic unc-59 mutation 35 .

Mammalian Cell Culture
Cells were cultured according to standard mammalian tissue culture protocols including testing
To generate ss-pCFJ1954-KDEL, the signal sequence of sel-1 was codon-optimized 53 and cloned into pCFJ1954 using around-the-world PCR with primers sel-1 ss flex F + sel-1 ss eft-3p R followed by a KLD reaction (NEB). A codon-optimized ZF1 domain from pie-1 and the . CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint KDEL sequence were then cloned into ss-pCFJ1954 using around-the-world PCR with primers coZF1 KDEL Stop attB2 F + coZF1 flex R followed by a KLD reaction.
To generate pIREShygro-Venus-mAID-LMNA, the LMNA open reading frame was amplified from a plasmid encoding GFP-Lamin A 54 and cloned onto the C-terminus of 3xHA-Venus-mAID 33 within a pIRES-hygro3 backbone (Clontech).

Worm transformation
FT205, WEH251, WEH399, WEH434, and WEH447 were made by biolistic transformation using a Bio-Rad PDS-1000, as described 55   The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint described 20,35 . For WEH251, Z-stacks were acquired for mCherry every 20 seconds or every minute. For strains WEH260 and WEH296, Z-stacks were acquired for mCherry every minute, as described 20 . For WEH399, Z-stacks were acquired for mKate2 and DIC every 30 seconds.
For WEH434, Z-stacks were captured for mCherry every 40 seconds. Embryos that arrested during imaging were excluded from analysis. Time-lapse series were analysed using Imaris

Progeny counts
Single L4 worms from wild type N2 and degron reporter strains were singled onto three 12well plates. The wells were photographed 3.75 days later on a Leica M80 with a Leica MC120 HD camera. Juvenile L2-L4 and adult progeny were counted using the Cell Counter in Fiji (NIH). For comparison of LMN-1 reporter strains, three L4 XA3502 worms or WEH251 with or without zif-1 RNAi treatment were put on one 60 mm plate and progeny were counted 3.75 days later. The number of worms with motility deficits (Unc) was reported as a percentage of the total worms counted on each plate. Wells where the mother died (n=10) or the worms clumped (n=12) were excluded from progeny counts.

Cell cycle timing
To compare the speed of development between control and ZF1-tagged strains, the time from The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint ABx, four ABxx or eight ABxxx cells. One embryo from FT205 strain was excluded because of cytokinesis defect.
Tracking H2B-labeled nuclei were tracked over time using the surface function of Imaris with thresholding to segment objects.

Quantification of corpse membrane breakdown
Corpse membrane topology was measured using a line scan across the 2PB phagosome in the WEH434 strain. A line with 3-pixel thickness was drawn through the middle of the phagosome using Fiji (NIH) and the mean profile intensity was measured.

Quantification of nuclei size
The widest cross-section was used to calculate the nuclear area prior to AB cell division. The edge of the P1 nuclear lamina was traced using Fiji (NIH) from fluorescent images of XA3502 and WEH251, as well as WEH251 fed with zif-1 RNAi.

Fluorescence intensity measurements
Mean fluorescence intensity of the mCh::PH::CTPD reporter (Fig. 3) was measured in a circle with an area of 0.5 µm 2 using ImageJ (NIH). Fluorescence intensity of the plasma membrane at the anterior side of the embryo near the first polar body was measured. Fluorescence intensity of the first polar body was measured as an internal control to correct for bleaching. Data are reported as the ratio of the fluorescence intensity of the plasma membrane to that of the polar body.
Mean fluorescence intensity of the ZF1::mCh::CHC-1 reporter (Fig. 4)  Mean fluorescence intensity of the LMN-1 reporter (Fig. 7) was measured in a circle with an area of 0.8 µm 2 using ImageJ (NIH) in AB and later in ABp and its daughter nuclei as well as The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint zif-1 RNAi ratio to observe changes due to ZIF-1-mediated degradation. A line was fit to three parts of the curve in Excel to calculate the slopes of the sharp drops during mitosis (from -1 to 2.7 min after the start of cytokinesis in the AB cell and from 11.3 to 13 minutes for ABx division) and the mild fluorescence loss during interphase (4 to 11 minutes).
For analysis of Venus-mAID-LMNA degradation (Fig. 8) Mean fluorescence intensity of the NMY-2::GFP::ZF1 reporter (Fig. 9) was measured in a circle with an area of 0.5 µm 2 using ImageJ (NIH), as described previously 35 . Midbody fluorescence was measured from contractile ring closure until the end of the time lapse series or until the midbody was not distinguishable from the cytoplasm. Fluorescence intensity of the first polar body was measured as an internal control. An exponential decay curve was fit to the polar body data using OriginPro (OriginLab) and used to correct for fluorescence loss due to photobleaching. Embryos were excluded if the P0 and AB midbodies were too close to each other (n=4) or if the polar body data did not fit an exponential decay function (n=1). Embryos who arrested development during time-lapse imaging were excluded from measurements (n=3).  The mean fluorescence of H2B reporters (Fig. S4b) was measured in a circle with an area of 2.6 µm 2 in the center of interphase nuclei from 2-to 15-cell stage embryos using Fiji (NIH). In older embryos where ZF1::mCh::H2B was no longer visible in AB nuclei, GFP::H2B was used to find the nucleus. Data is reported as a ratio of the mean fluorescence of the anterior AB lineage nuclei to the posterior P lineage nuclei. Images were excluded when AB or P nuclei had condensed mitotic chromosomes (n=29).
Mean fluorescence intensity of LMN-1 reporters for Fig. S4d was measured using a 3 µm line two pixels in width using Fiji (NIH). The line was drawn at the brightest region of the nuclear membrane from images of 2-to 15-cell stage embryos. The intensity was measured in anterior AB cells and posterior P cells, except when P cells were dividing, in which case P sister cells, including E at the 7-cell stage (n=1) or C at the 14-cell stage (n=1) were measured. In older embryos where ZF1-tagged LMN-1was no longer visible in AB nuclei, YFP::LMN-1 was used to trace the nucleus. Embryos were excluded during NEBD of ABx (n=5) or when the nuclei morphology was extremely malformed (n=4) or when the cell identity could not be determined (n=1).
Mean fluorescence intensity of NMY-2 reporters (Fig. S4f) was measured in a circle with an area of 1 µm 2 in midbodies after abscission in 2-to 15-cell embryos using Fiji (NIH). Data is presented as a ratio of the fluorescence intensity of anterior midbodies to posterior midbodies.
In older embryos where ZF1-tagged NMY-2 was no longer visible in AB nuclei, NMY-2::mCh was used to locate the midbody. Images were excluded when midbodies were out of focus (n=3) or endocytosed (n=20) or when the cell identity could not be determined (n=1).

Fluorescence recovery after photobleaching
Fluorescence recovery after photobleaching (FRAP) was conducted using a Leica SP8 confocal with a HCX PL APO CS 40.0x1.25-NA oil objective with PMT detectors. Half of the ABp nucleus was bleached using the 560 nm laser line. Images were acquired before and after bleaching with intervals of 2 seconds (5 times), 5 seconds (15 times), and 10 seconds (10 times). The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint (n=3). In 2 movies focus was lost during the last two minutes. Those time points were not included in measurements.

Image processing
For clarity, images were rotated, colorized and the intensity was adjusted using Adobe Photoshop. All images show a single optical section (Z), except for Fig. 5, Fig. 7, Fig. 10, Fig.   S2, and Fig. S5. In Fig. 5, six Zs with 1.2 µm steps were maximum projected using Leica LAS X software. In Fig. 7, three Zs with 1.2 µm steps were maximum projected using Leica LAS X. In Fig. 10, two Zs with 1.2 µm steps were maximum projected using Fiji (NIH). In Fig. S2, images where maximum projected to span a region of 2.5 µm using Fiji (NIH). In Fig. S4a, four Zs with 1.2 µm steps were maximum projected using Fiji (NIH). In Fig. S4c, two Zs with 1.2 µm steps were maximum projected using Fiji (NIH). In Fig. S4e, six Zs with 1.2 µm steps were maximum projected using Fiji (NIH). Fig. 5c, 5f, and videos were rotated, colorized, and the intensity was adjusted using Imaris. Several Zs were maximum projected in Imaris for videos. For the left video in video 4, the brightness was adjusted in each frame using Photoshop to compensate for photobleaching.

Statistical evaluation
Student's one-tailed t-test was used to test statistical significance, except Fisher's exact test and a chi-squared test were used to test statistical significance of categorical data. A Bonferroni correction was used to adjust for multiple comparisons. Prism 6.0 (Graphpad) was used for statistics and to create graphs for Fig. 8. Venus-mAID-LMNA degradation curves represent single cell data of the indicated number of cells. Differences in average Venus-mAID-LMNA degradation velocities were analysed for significance using an unpaired multi-comparison Kruskal-Wallis test with Dunn's statistical hypothesis testing presenting multiplicity-adjusted P values. All experiments are representative of at least three independent repeats unless otherwise stated. No randomization or blinding was used in this study.

Data Availability
The source data underlying Figs 3c, 4e, 7g-h, 8f, 8g, 8h, 9g, 10c and Supplementary Figs 1a-f,   3, 4b, 4d, 4f are provided as a Source Data file. Full data sets generated during and/or analysed during the current study are available from the corresponding author on request.
. CC-BY-NC 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint           The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint         The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/442103 doi: bioRxiv preprint